This proposal is devoted to the study of microsomal hydroxylases from the testis and ovary. These enzymes catalyze the conversion of steroid precursors to estrogens and androgens. The initial studies will be directed toward identification of the microsomal components involved in these reactions in addition to those already delineated by previous studies. Specifically, the role of cytochrome b5, phospholipids, and a carrier protein will be evaluated. Subsequently, studies aimed at determining the nature of the P-450 binding site will be carried out by estimating binding constants of varying steroids through spectral analysis. The P-450 content of testis will be determined to resolve the problem of whether hydroxylase activities are catalyzed by single or multiple species of this cytochrome. Induction of P-450 will be effected by gonadotrophins to determine if these hormones elicit a new species of P-450 to explain the observed changes in steroid hormone synthesis. A comparison of hydroxylases from other steroid metabolizing tissues including adrenal, liver, and placenta will be conducted to gain insight into the factors conferring enzymic specificity. Control of hydroxylase activity by precursors and products will be evaluated. The changes in ovarian enzymic activity will be evaluated to explain the changes in blood hormone concentrations observed in the human menstrual cycle. The nature of the activated oxygen involved in cleavage of the C-C bond of the steroid alpha-ketols will be investigated using systems developed in studies on liver hydroxylases.